Note: Descriptions are shown in the official language in which they were submitted.
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Liquid biological antifungal product containing the Pythium oligandrum
microorganism and
method of production
Technical Field
The invention concerns a liquid biological antifungal product containing the
Pythium
oligandrum microorganism.
The invention also concerns the method of producing this product.
Background of the Invention
Chemical antifungal products have in recent decades aroused rising fears from
the
perspective of their negative influence on the environment, and indeed the
user. Nonetheless, it can
be said that the replacement of these with effective biological products based
on microbes that are
able to eliminate molds in a targeted and considerate way has not proceeded to
the desired extent.
Only 14 microbial types are currently registered for this purpose in the
countries of the EU, this in
spite of very intensive research, as stressed in a recent article by Gerbore
et al from 2014 [1].
The practical application of a biological product which uses the antifungal
effects of the
Pythium oligandrum microorganism has been known since the 1970s, in that we
can here refer to
the classic works of Dreschler [2] from 1943 and Deacon [3] from 1976
dedicated to the biology of
this microorganism and describing its mycoparasitic properties. It is from
that time that we have
patent US 4,259,317 [4], authors Veseljf and Hejdanek, priority date 5.7.1978,
dedicated to dry
powder products containing oospores of Pythium oligandrum Dreschler, for
protection against
mold-related diseases emanating from sugar beet shoots having an content of
oospores of a
minimum 1 million oospores per gram of product, whereby a quantity of between
50 and 500
million oospores per gram of product is stated as the optimum level. As an
applicable alternative to
dry powder products, the authors of this invention accept a liquid suspension
of oospores, but
specify a number of disadvantages for the application which they prepared, in
particular the
instability of such products, as limiting their practical application to
around 2 weeks. After a longer
period of time, there is an unacceptable reduction in the emergence of
oospores, which is essential
for application of the mycoparasitic protection mechanism, in consequence of
autolysis or
contamination by hyperparasites. As far as the source of individual strains of
Pythium oligundrum
is concerned, authors Baker and Lifshitz pointed in their patent US 4,574,083
[5] to the possibility
of obtaining regionally suitable strains through isolation from the soil
according to the standard
published method. This procedure has since been vindicated and strains from
different
geographical locations are now available in public collections. The following
biological criteria
were mainly applied at that time to identify isolates: speed of growth,
optimum growth temperature
¨ usually around 30 C, and morphology of individual reproductive forms of the
sexual and asexual
cycle. From this perspective, the patent which followed can be deemed ground-
breaking; in this,
inventor Martin, US 5,961,971 [6], introduces the molecular characterization
of individual isolates
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based on the precise sequential protocol of critical genes. Individual
formulations and methods of
applying the Pythium oligandrum microorganism in field conditions are
presented in detail in the
same patent document, whereby liquid formulations following growth on cheap
media based on
industrial waste materials and powder formulations prepared by the
encapsulation of biomass dried
after fermentation to become alginates or in the form of a dried and ground
product are mentioned
as the preferred formulations. Formulations in the form of wettable powders,
standard powders,
emulsified oils and granules are among the other possible formulations. Patent
CZ 303 908 and the
corresponding EP 2 503 892 [7], BIOPREPARATY, spot. s.r.o., from 2009, focuses
on the post-
harvest protection of crops with the use of the Pythium oligandrum
microorganism. In the
technological section, it concentrates on the culturing of this microorganism
in standard fermenters
on liquid media with cereal content, without the addition of other nutrients.
The possibility is
accentuated of obtaining a pure concentrate of oospores without the presence
of exhausted media or
the remains of mycelium, which are removed during drying in a spray drying
oven at the late stages
of the production process. Knowledge of the preparation of oospores of
organisms other than
Pythium oligandrum has been published to a limited extent. In patent
application CN 102 807
957A [8], Liu X. et al, from 2012, the preparation of emerging oospores of the
Phytophthora
capsici microorganism is described in order to monitor resistance to various
drugs and for genetic
research. Liquid products for the biological protection of plants are
specified in patent application
US 2014/0212387 [9], Liith, from 2014, although these are exclusively liquid
products that use the
polyethers of modified trisiloxanes as solvents.
The preparation and stabilization of oospores and other reproductive forms of
microscopic
Pythium oligandrum oomycetes is also discussed to a certain extent in non-
patent literature. In
1990, McQuilken et al [10] published a simple and reproducible method for the
preparation of
oospores in a liquid culture with the use of molasses as an economically
affordable substrate.
However, the issue of the ideal method of maintaining prepared oospores has
not been
satisfactorily resolved in either patent or non-patent literature. In 1992,
McQuilken et al [1 1 ]
observed the influence of different osmotics, such as glycerol, NaCI and KCI,
on the speed of
radial extension of hyphae and the emergence of oospores of the Pythium
oligandrum oomycete by
way of a comparison with the osmotic pressure presented by a soil extract at -
0.5 MPa. The
addition of osmolytes reduced the speed of radial extension, but had no
influence on emergence to
a value of around -2.0 MPa. The authors conclude that good effectiveness of
the applied oospores
can be expected under normally-occurring osmotic conditions in the places of
application on the
condition that conditions are met from the perspective of optimum temperature,
generally 30 C,
and acidity of pH 6.0 to 7.5. From the perspective of the storability of the
oospores obtained, their
high effectiveness for a period of up to 379 days when maintaining a normally
prepared suspension
in distilled water in the refrigerator at 4 C is mentioned by authors
Takenaka and lshikawa [12] of
2013.
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According to experts, the main obstacle to greater spread of biological
defense products is
quite clearly the dependence of effectiveness on specific conditions of
application and practical
problems with maintaining and preparing biological products to protect plants
in everyday
agricultural work. For this reason, resolving practical problems associated
with the simple and
effective storage and application of Pythium oligandrum oospores is crucial to
the commercial
success of biological products. in order to resolve this problem, it would be
desirable to develop
means of biological protection in liquid state, with high long-term stability
even under standard
storage conditions. However, the solutions published so far suffer from a
number of disadvantages
that have not been successfully eliminated even after years of intensive
effort. The majority of
procedures involved in the laboratory preparation of biological preparations
to protect plants are
complicated and only manageable at laboratory level, which significantly
restricts the use of such
procedures for industrial production. The preferred method of application
until now, in the form of
a wettable powder, which was applied in products manufactured by Czech
biotechnological
company Biopreparaty, spol s.r.o. (Polyversumg, Polygandron, BIOREPELO) and
sold
worldwide, is not without its own complications from the perspective of
targeting defensive
intervention and protecting the users. Until now, there has been no simple,
well-tested and
consistently-defined procedure published for a formulation with sufficient
storability of prepared
reproductive forms of the Pythium oligandrum microorganism, or of oospores, in
a simple form
that can be used easily in industry.
Summary of the Invention
The specified disadvantages of the biological antifungal products applied
until now are
eliminated or restricted under this invention, whose essence is a liquid
biological antifungal product
containing the Pythium oligandrum microorganism according to independent claim
1, including a
stabilized suspension of Pythium oligandrum, and containing 0.05 to 10.0 A
weight culturing
biomass of the Pythium oligandrum microorganism with content of cultivation
medium, cell forms
of this microorganism and substances produced by this microorganism; and 90.0
to 99.95 %
weight stabilizer; whereby the number of dormant oospores in 1 ml of this
liquid biological
antifungal product required in advance ranges from 1 x 103 to 2 x 10 following
normal
standardization.
The essence of the liquid biological antifungal product containing the Pythium
oligandrum microorganism according to the 21'd independent claim of this
invention is based on the
fact that it includes a stabilized suspension of Pythium oligandrum and
contains: 0.05 to 10.0 %
weight culturing biomass of the Pythium oligandrum microorganism with content
of cultivation
medium, cell forms of this microorganism and substances produced by this
microorganism; 79.77
to 99.95 % weight stabilizer; the remainder, to 100 % weight, at least one
modifying/application
substance from a group including filling, aroma, vitamin E; whereby the number
of dormant
oospores in 1 ml of this liquid biological antifungal product required in
advance ranges from
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2.5 x 104 to 1.0 x 106 following normal standardization.
Preferable is the Pythium oligandrum Dreschler ATTC 38472 strain of the
Pythium
oligandrum microorganism, which was deposited in the Czech Collection of
Microorganisms
(CCM) at Masaryk University in Brno under the appellation of Pythium
oligandrum Ml.
The main advantage of this invention is a new liquid biological antifungal
product that is
suitable for long-term usage and achievement of its long-term and significant
stabilization effect,
whereby the stabilizers used are affordable and provide a further advantage in
protecting the
contained biomass of the Pythium oligandrum microorganism from microbial
contamination. The
claimed content of dormant cells of the microorganism specified above in the
form of oospores
provides a practically tested and economically affordable scope, which
facilitates simple dosing for
a wide range of required applications. It can use different types of biomass,
processed using a
suitable preparation. The lower concentration of dormant oospores, for example
in tens or hundreds
of dormant oospores, also brings results, although the effectiveness of this
product is lower. The
pre-determined number of dormant oospores can be regulated, if required, by
normal
standardization, i.e. by diluting or concentrating to the required value.
It is advantageous when the stabilizer contains at least one component from a
group
including water, salt solution, oil or solutions of osmolytes, which might be
polyol solution, of
saccharide or saccharide alcohol type, or salt solutions. Polyol solutions may
be chosen from a
group containing metabolizable or unmetabolizable solutions of various
oligosaccharides
containing a maximum of 10 saccharide units and a minimum of 2 saccharide
units, such as
maltodecaose or maltononaose or maltooctaose or maltohelpaose or maltohexaose
or
maltopentaose or stachyose or rafinose or sucrose or sucralose, or branched
saccharides may be
used.
In the ideal execution, the stabilizer is water in a quantity of, for example,
30.0 to 99.9 %
weight, or salt solution in a quantity of, for example, 99.9 % weight, or an
osmolyte in the form of
sucrose in a quantity of, for example, 60.0 to 64.95 % weight, or at least one
oil selected from a
group including paraffin oil, mineral oil, glycerol, sunflower oil, in a
quantity of, for example.
79.77 to 99.9 % weight.
The liquid biological antifungal product may contain other modifying or
application
substances for a specific area of use, such as a slow-release biodegradable
matrix such as a carrier
of oospores of the Pythium oligandrum microorganism, for example polyvinyl
alcohol, in a
quantity, for example, of 16.0 % weight; or filling based on silicon oxide in
a quantity of, for
example, 16.0 % weight; or other application/modifying substances, such as
sage or mint aroma in
a quantity of, for example, 0.96 % weight, or vitamin E in a quantity of, for
example, 0.4 % weight.
The use of such application/modifying substances depends on the application of
the
biological antifungal product.
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Consequently, a stabilized suspension of oospores of the Pythium oligandrum
microorganism might use a wide range of stabilizing agents of a hydrophilic or
hydrophobic nature,
such as common water or oil or solutions of different affordable osmolytes.
Osmolytes can be perfectly adapted, through their chemical nature, to the
required
applications, since it has been shown that osmolytes might be of an ion
nature, such as solutions of
different salts, or of a non-ion nature, such as polyol solutions. The fact
that polyols might be
represented by, for example, metabolizable or unmetabolizable solutions of
different defined
oligosaccharides is beneficial for a number of applications, because mixtures
of suitable natural or
synthetic substances of the required properties can be used for this purpose.
A commonly available
substance such as sucrose can also be used to our advantage, but if required
even commonly
available unmetabolizable analogues can be used. The composition of liquid
suspensions is
compatible with a wide range of other substances which modify their utility
properties or their
applications in the form of antioxidant substances, natural activators and
vitamins, for example
silicon oxide as a filling, vitamin E acetate and other substances.
It was discovered, somewhat surprisingly, that if the cell percentage produced
by solid or
liquid culturing following the end of preparation is put in the form of a
suspension, or concentrated
suspension, the reproductive forms of the Pythium oligandrum microorganism
retain the original
emergence in such a suspension for at least six months if normally stored at
laboratory temperature.
This result becomes even more prominent when we consider the irreproducible
and variable results
presented in the published literature. It is stated in literature, for
example, that a suspension of
oospores prepared in distilled water and stored for a period of 188 or 384
days retained the original
emergence when stored in a refrigerator at 4 C. Nonetheless, the entirely
absent description of the
method of preparing such described preparations makes it almost impossible to
evaluate their
scientific validity, not to mention practical applicability, when a storage
temperature of 4 C might
not even be available under working conditions on farms.
The liquid biological antifungal product containing the Pythium oligandrum
microorganism is obtained using the method of production according to this
invention, the essence
of which lies in the fact that a liquid medium containing extract of cereals,
cane molasses and other
essential nutrients is sterilized in a steam sterilizer by way of the aerobic
culturing of the Pvihium
oligandrum oomycete in liquid phase. After cooling, it is inoculated with one
of the selected strains
of Pythiurn oligandrum. The biomass is taken and processed after the end of
culturing, which takes
several days, preferentially 13. After the end of the culturing stage of
Pythium oligandrum in liquid
phase, the biomass is homogenized with the liquid medium so that a minimum of
95 % of the
particles in the suspension are between 0.050 and 0.300 mm, preferentially
0.125 mm. The
homogeneous suspension characterized by the number of oospores is concentrated
or diluted
according to the number of oospores in the solution to the pre-determined
concentration of
oospores in the biological antifungal product. The suspension is
preferentially filtered after the
stage of homogenization and concentration or dilution.
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An aqueous suspension is stabilized during homogenization with an additive of
osmolyte and
is stored in large, sterile containers at temperatures of lower than 8 C.
In the case of an anhydrous suspension of the Pythium oligandrum
microorganism, the
material obtained after the end of the culturing stage on a solid substrate or
at the liquid phase is
homogenized until reaching a value of a minimum 95 % of the particles in the
suspension between
0.050 and 0.300 mm, preferentially 0.125 mm. The obtained homogenous
suspension characterized
by the number of oospores is centrifuged. After the removal of supernatant,
the centrifuged
material is supplemented with oil to achieve the pre-determined concentration
of oospores for the
production of the biological antifungal product in such a quantity that the
resulting concentration of
oospores in the biological antifungal product is correct to the desired
concentration and the
material obtained in this way is subsequently re-suspended by homogenization.
The resolution presented would appear to be suitable even from the perspective
of
significant simplification of the biotechnological process of preparing the
Pythium oligandrum
microorganism and thus the significant reduction in running costs for the
manufacturers of such
products. IFiltration or separation of the cultivated biomass from the
production medium, its drying
and subsequent grinding to the required size of particles entails several
processes that are relatively
demanding on energy and technology, difficult to control and susceptible to
the possibility of
contamination, which could, as an end result, significantly damage the
resulting product, if not
carried out under precisely-defined and industrially demanding conditions. By
contrast, the
technologically simple suspension of the biomass obtained in dense and viscose
solutions of
osmolytes results in a very stable product that is immediately suitable for a
range of practical
applications. From the practical perspective, it is a significant advantage
that the prepared
suspension of the Pythium oligandrum microorganism can be transferred from
culture dishes or
fermenters almost without waste, which minimizes the costs of their cleaning
and maintenance and
also minimizes the possibility of product leaking into the work environment of
the production
premises. Moreover, the very fact that the product obtained in this way is
stable over the long-term,
even during storage at normal temperatures, also significantly reduces running
costs and makes it
easier to handle the product.
It was also discovered, surprisingly, that apart from the stabilizing effect
of concentrated
solutions of osmolytes, the conserving effect of such solutions, which is well-
known from
production and common practice, is significant in the specific case of the
suspensions of oospores
of the microscopic oomycete Pythium oligandrum which we examined. A product
stored in this
way is therefore protected from contamination by undesirable microorganisms,
which, even in the
very unlikely case of penetration of such stabilized suspensions, are unable
to reproduce in the
proposed chemical environment and cannot therefore contaminate the end
product. This advantage
is fundamental in current production practice, particularly in light of the
ever stricter requirements
on the level of accidental contamination in pesticides, biocides and cosmetic
products.
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In addition to the role of stabilizer and the role of conserving substance,
osmolyte solutions
also play the role of adhesive, a fact which can be practically applied in
many applications, whether
in the form of aqueous solutions after the dilution of stabilized suspensions
of oospores to the
optimum concentration with application by soaking or for application by gentle
dispersion of
microscopic particles (so-called misting). In all of the examples of
application specified above,
polyol solutions therefore aid effective adhesion and subsequent even
application to the treated
areas, plants or crops.
Last, but not least, it must be mentioned that in the case of using
metabolizable osmolytes,
these substances might serve as a nutrient which is important during
colonization and settlement of
the target places directly at the places of application, whereby this is a
significant nutrient
particularly during the first stage connected with the transfer of oospores to
myceliar form. They
can subsequently take on the role of nutrients important for the long-term
growth and preservation
of the microscopic oomycete Pythiurn oligandrum in the place of application of
a nutrient of more
complex nature, represented by imperfectly fermented substrate used during
biotechnological
growth and reproduction of oospores during biotechnological culturing.
The specific content of oospores and other reproductive forms of microscopic
oomycetes in
suspensions according to the resolution presented is comparable with or higher
than previously-
tested and sold loose products, which is important from the perspective of the
logistics and
economics of distribution of a product made in this way. However, the new
formulation also
makes it possible to use lower concentrations in comparison with the previous
resolution.
We have actually seen a significant reduction in composition complexity in the
new liquid
biological antifungal product in connection with the technological procedure
used, without this
being linked to any limitation in the function or effectiveness of the liquid
product formulated in
this way. As far as the cell and particle composition of the newly-proposed
product is concerned,
this almost entirely entails stabilized oospores with a very low percentage of
mycelial fragments,
ground millet and other components. These components obviously do not sediment
in the dense
environment created by concentrated osmoles, which surprisingly guarantees
good homogeneity of
the product, incomparably better than in the case of wettable powders applied
in the form of
aqueous suspensions, when the preparation was very heterogeneous from the
perspective of the size
of the particles therein. This particularly applies to the particles of
silicon oxide contained in the
products made until now; by contrast, silicon oxide is absent in most cases in
the resolution
according to the submitted invention (with the exception of toothpaste, where
it is used as an
abrasive). Consequently, from the perspective of the needs of practical
applications, there is no
clogging of filters and other technological equipment during the application
of the product.
If the presence of a lower quantity of metabolizable osmolytes is detrimental
to a certain
application, these can easily be replaced with chemically similar
unmetabolizable osmolytes
without affecting in any way the most important properties of the product.
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If it is more appropriate from the technological perspective to dry the
product obtained,
freeze-drying (Iyophilization) would appear to be suitable here, in apparatus
with a high vacuum
value, since the use of concentrated solutions of osmolytes as
cryopreservatives and protective
substances during lyophilization is well-known and widely-used. By way of
example, we refer to
the osmolytes widely used as protective cryopreservatives in a number of
sensitive applications,
including the cryopreservation of human oocytes described, for example, in an
article by Wright
[15] from 2004.
A considerable advantage of the products obtained according to the submitted
invention is
that such products are suitable for all applications in which it would appear
beneficial to use the
anti-mold, mycoparasitic, elicitor and growth properties of the Pythium
oligandrum
microorganism. In particular, therefore, the use of this product as a plant
biofungicide, for human
and veterinary use of the Pythium oligandrum microorganism, for the
destruction of biofilms and
the removal of dysbiosis occurring in the case of various medical diagnoses
and also for civic
protection from molds occurring in the living and/or working environment.
Depending on the
matrices used in the liquid suspension concentrate, it can be used as a
protective spray of plants in
the presence of a degradable biopolymer, which acts as a protective factor and
as a gradually
degradable matrix, meaning as a "carrier" of Polyversum material with oospores
of the active
microorganism Pythium oligandrum.
Protection against molds, funguses, pathogenic bacteria and yeasts with the
use of a
stabilized, concentrated suspension of oospores of the Pythium oligandrum
microorganism,
consisting of the application of such stabilized, concentrated suspension, in
undiluted or diluted
form, to an area affected by mold or fungus or pathogenic bacteria or
pathogenic yeasts or
microbial dysbiosis, makes it possible to define a wide scope of applications
for the stabilized
liquid concentrate.
In the sphere of plant protection, the composition can be used to provide
protection against
molds, funguses or pathogenic bacteria and yeasts such that the stabilized,
concentrated suspension,
in diluted or undiluted form, is applied to plants or their surroundings or
seeds or crops or fruits
either before harvest or after harvest, for example in warehouses; this
includes application in the
form of aerosols, self-soluble capsules containing concentrated suspension,
additional fertilization
at the bottom of the plant, soaking crops, spraying crops, seed treatment,
misting crops, fertilizing
and additional fertilizing as a component part of comprehensive fertilizers
and application in the
form of hydroponics.
When protecting the oral cavity, the stabilized, concentrated suspension in
diluted or
undiluted form can be applied to a place affected by oral plaque or
periodontal disease, the
occurrence of infected, non-healing wounds, in particular among diabetics, the
occurrence of
dermatophytal or yeast-related infectious diseases and the occurrence of other
symptoms connected
with microbial dysbiosis of the skin and human surface membranes.
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For use in civic protection, the stabilized, concentrated suspension, in
diluted or undiluted
form, can be applied to places on walls and in masonry affected by the
occurrence of fungus. Other
innovative areas of use include antifungal protection of cooling equipment,
air-conditioning, areas
affected by flooding and deluge and other places with frequent occurrence of
funguses targeted by
the Pythium oligandrum microorganism.
Brief Description of the Drawings
The invention is further described in detail in exemplary embodiments and
explained in
more detail in the appended schematic drawings.
Figure 1A, 1B, 1C elucidate in more detail a comparison of the speed of growth
of the
phytopathogenic fungus Fusarium graminearum without the presence of the
PI/Miran oligandrum
microorganism and when that microorganism is present.
They specifically show:
Figure 1A the growth of a colony of the phytopathogenic fungus Fusarium
graminearum on its
own (square) and with simultaneous inoculation by Pythium oligandrum from
dormant oospores
and from liquid and solid culturing (stars and triangles);
Figure 1B photograph of Petri dishes on which the Pythium oligandrum
microorganism from
dormant oospores has been inoculated on the left-hand side and the
phytopathogenic fungus
Fusarium graminearum (upper row) on the right-hand side and on control dishes
only Fusarium
graminearum fungus was inoculated, in that growth was recorded after 4 days;
Figure 1C photograph of Petri dishes with simultaneous inoculation
microorganism of Pythium
oligandrum from dormant oospores of the phytopatogenic fungus Fusarium
graminearum, where
growth was recorded after 11 days.
Figure 2A, 2B, 2C show the results of laboratory experiments of the growth of
wheat in the
presence of the phytopathogenic fungus Fusarium graminearum, the Pythium
oligandrum
microorganism and a combination of these two microorganisms.
They specifically show:
Figure 2A the Erlenmeyer flasks in which wheat grew on agar soil in the
presence of only the
phytopathogenic fungus Fusarium graminearum, the little white spots, recorded
after 20 days of
inoculation at the shoot of the wheat;
Figure 2B the Erlenmeyer flasks in which wheat grew on agar soil in the
presence of both the
phytopathogenic fungus Fusari urn graminearum and the Pythium oligandrum
microorganism; the
flasks were recorded after 20 days of simultaneous inoculation at the shoot of
the wheat: and
Figure 2C the Erlenmeyer flasks in which wheat grew on agar soil in the
presence of only the
Pythium oligandrum microorganism; the flasks were photographed after 20 days
of inoculation of
this fungus at the shoot of the wheat.
Figure 3A, 3B, 3C, 3D, 3E show the application of an antifungal product for
plant
protection according to exemplary embodiment 1 and subsequent analysis of the
population
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dynamic of the Pythium ohgandrum microorganism observed in a field experiment
on wheat and
the adjacent soil, including reaction to the presence of the fungus monitored
by gene expression
and genetic observation of the level of fungal contamination.
They specifically show:
Figure 3A the content of the Pythium oligandrurn microorganism determined by
genetic test on
plants (light-gray column) and in the surrounding, adjacent soil (dark-gray
column) at different
times following application;
Figure 3B the development of temperatures measured using an infrared
thermometer right in the
field at the time of sampling (light-gray column) and a temperature record
obtained from the local
meteorological station (dark-gray column);
Figure 3C the course of gene expression for cellulase (POCELL), endo- -
glucanase (POENDO)
and a structural protein rich in serine and threonine (POSTRU) in individual
parts of the plant and
in surrounding soil (So - soil, R - root, St - stem, L - leaf) observed at
time 6 to 120 hours, whereby
the level of gene expression was standardized to the expression of the
constitutive gene -tubulin
(POTUBU) and is expressed without the application of the product according to
the cited
methodology [12];
Figure 3D the level of fungal contamination in identical samples as in panel C
measured using
quantitative PCR with general amplification primers for the amplification of
fungus.
Figure 3E a correlation graph monitoring the correlation between the level of
gene expression for
cellulase (POCELL) and the level of fungal contamination.
Figure 4A, 4B, 4C, 4D show a test of the effectiveness of toothpastes
according to
exemplary embodiment 4 from the, perspective of their ability to eliminate
biofilms artificially
created on ceramic hydroxylapatite plates, with the use of saliva from a
healthy individual in the
upper part of the figure and the use of saliva from an individual suffering
from periodontal disease
in the lower part of the figure.
They specifically show:
Figure 4A a photograph of a six-hole plate with the use of saliva from a
healthy individual after the
release of rinsed biofilms in the solution and the removal of ceramic
hydroxylapatite plates which
were previously cleaned with a brush without paste, with Odol toothpaste, with
Enzycal toothpaste,
with paste containing glycerol according to exemplary embodiment 4 without the
addition of the
Pythium oligandrum microorganism, with paste containing glycerol according to
exemplary
embodiment 4 without the addition of silicon oxide and full paste containing
glycerol according to
exemplary embodiment 4;
Figure 4B a quantitative evaluation of the content of the biofilm by measuring
the intensity of
coloring in individual holes of Figure lA on a plate spectrophotometer at a
wavelength of 562 nm;
Figure 4C a photograph of a six-hole plate with the use of saliva from an
individual with
periodontal disease after the release of rinsed biofilms in the solution and
the removal of ceramic
hydroxylapatite plates which were previously cleaned with a brush without
paste, with Odoi
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toothpaste, with Enzycal toothpaste, with paste containing glycerol according
to exemplary
embodiment 4 without the addition of the Pythium oligandrum microorganism,
with paste
containing glycerol according to exemplary embodiment 4 without the addition
of silicon oxide and
full paste containing glycerol according to exemplary embodiment 4; and
Figure 4D a quantitative evaluation of the content of the biofilm by measuring
the intensity of
coloring in individual holes of Figure IC on a plate spectrophotometer at a
wavelength of 562 nm.
Exemplary Embodiments of the Invention
The liquid biological antifungal product contains the Pythium oligandrum
microorganism and
in all exemplary embodiments the Pythium oligandrum microorganism is of the
Pythiurn
oligandrum Dreschler ATTC 38472 strain, which was deposited in the Czech
Collection of
Microorganisms (CCM) at Masaryk University in Brno, under appellation Pythium
oligandrum
MI, and it is under this appellation that it is presented hereunder in
exemplary embodiments.
Below is the Sequence Protocol of this microorganism according to ST25_PCT:
<160> 2
<210> 1
<211> 759
<212> DNA
<213> Pythium oligandrum strain M1
<221> ITS rRNA gene
<400> 1
atcattacca cacctaaaaa ctttccacgt gaaccgttat aactatgttc tgtgcttcgt 60
cgcaagactt gaggctgaac gaaggtgagt ctgcgtctat tttggatgcg gatttgctga 120
tgttatttta aacacctatt acttaatact gaactatact ccgaatacga aagtttttgg 180
ttttaacaat taacaacttt cagcagtgga tgtctaggct cgcacatcga tgaagaacgc 240
tgcgaactgc gatacgtaat gcgaattgca gaattcagtg agtcatcgaa attttgaacg 300
catattgcac tttcgggtta tgcctggaag tatgcctgta tcagtgtccg tacatcaaac 360
ttgcctttct ttttttgtgt agtcaaaatt agagatggca gaatgtgagg tgtctcgcgc 420
tgtcttttta aagatggttc gagtcccttt aaatgtacgt tgattctttc ttgtgtctgc 480
gaattgcgat gctatgctct ttgtgatcgg tttagattgc tttgcgctgg tgggcgactt 540
cggttaggac atatggaagc aacctcaatt ggcggtatgt tcggctttgc ctgacgttaa 600
gctaagcgag tgtagttttc tgtcttttcc ttgaggtgta cctgtcgtgt gtgaggttga 660
tttaggctat atggttgctt ggttgtgtgg tttagcgttt tcagacgcct gcttcggtag 720
gtaaaggaga caacaccaat ttgggactga gagtttact 759
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<210> 2
<211> 683
<212> DNA
<213> Pythium oligandrum strain M1
<221> Mitochondrial COXII cytochrome oxidase
<400> 2
atggaaggta ttattaactt tcatcatgat ttagtatttt ttttaattat tgtgactgtt 60
tttgtttgtt ggttattatt tagagtaatc gtattattcg atgaaaaaaa aaacccaata 120
cctqctacat ttgtacatgg agcaactatt gaaattattt ggacaacaat tccagcatta 180
attttattaa ccgtagcagt tccatctttt gctttattat attcaatgga tgaaattatt 240
gatccaatta taactttaaa agtaataggt agtcaatggt actggagtLa tgaatattct 300
gataatttag aatttgcaga tgaaccttta atttttgata gttacatggt tcaagataat 360
gacttagaaa taggacaatt taggttatta gaagtagaca accgtgttgt tgtaccaact 420
aatagccata ttagagtttt aataacagct tctgacgttt tacattcatg ggctataccc 480
tctttaggtt taaaattaga tgcttgtcca ggtcgtttaa atcaaacttc aatgtttatt 540
aaaagagaag gtgtatttta cggtcaatgt agtgaaatat gtggtataaa tcatggtttt 600
atgccaatag ttgttgaagc agtttcatta gaagattatt tagtttggtt aaaaaacaaa 660
ttaattttga ttttaatgta taa 683
Example 1
Liquid biological antifungal product intended for spraying crops with high
percentage by
weight of biomass
Preparation:
Biomass obtained through culturing of the Pythiurn oligandrum M1 microorganism
on a
solid substrate was mixed in a homogenizer vessel (industrial mixer) with
demineralized water and
homogenized. Homogenization proceeded at 3,000 ¨ 5,000 revolutions per minute
for a period of 3
minutes. Subsequently, an osmolyte (sucrose) was added to the initial,
undiluted suspension in a
quantity corresponding to the resultant concentration by weight of 60 % weight
and to the required
number of oospores. The suspension with osmolyte (sucrose) was thereafter
homogenized in a
mixer for 1 minute at 2,000 revolutions per minute. The suspension was then
stored in sterile,
stainless steel tanks at a temperature of under 8 C.
It was more beneficial to prepare the liquid biological antifungal product by
combining the
steps of homogenization in demineralized water and subsequent addition of
osmolyte (sucrose) in
one, in such a way that the biomass obtained through solid culturing was
homogenized in the vessel
of an industrial homogenizer in an osmolyte solution (65 % sucrose solution).
Homogenization
proceeded at 3,000 ¨ 5,000 revolutions per minute for a period of 3 minutes.
After determining the
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number of oospores in the initial suspension, this suspension was diluted with
osmolyte (65 %
sucrose solution) in the standard way such that the required concentration of
oospores in the
product was achieved. The suspension was then stored in sterile, stainless
steel tanks at a
temperature of under 8 C.
The number of dormant oospores was determined microscopically with the use of
a
Sedgewick-Rafter counting chamber and stability was measured based on
microscopic observation
of the number of viable oospores using the plasmolytic method, in accordance
with the publication
by Etxeberria et al [13] from 2011. If the number of viable oospores did not
fall below 90% of the
initial value, the product was marked as stable.
Composition of the product:
Sucrose as stabilizer 60
% weight
Water as stabilizer 30
% weight
Culturing biomass
10% weight
Number of dormant oospores of the Pythium oligandrum microorganism 5
x 105 per 1 ml
Properties:
Stability: 6 months at a temperature of up to 25 C.
Example 2
Liquid biological antifungal product intended for spraying crops with low
percentage by
weight of biomass
Preparation:
Pythium oligandrum biomass obtained by liquid cultivation was mixed in the
vessel of a
homogenizer (industrial mixer) with osmolyte (sucrose) and homogenized in such
a way that the
required number of oospores and concentration of osmolyte (sucrose) were
achieved.
Homogenization proceeded at a speed of 3,000 ¨ 5,000 revolutions per minute
for a period of 3
minutes. The suspension was then stored in sterile, stainless steel tanks at a
temperature of under 8
C. The number of dormant oospores was determined microscopically with the use
of a Sedgewick-
Rafter counting chamber and stability was measured based on microscopic
observation of the
number of viable oospores using the plasmolytic method, in accordance with the
publication by
Etxeberria et al [13] from 2011. If the number of viable oospores did not fall
below 90% of the
initial value, the product was marked as stable.
Composition:
Sucrose as stabilizer 64
% weight
Water as stabilizer
35.9 % weight
Culturing biomass 0.1 %
weight
Number of dormant oospores of the Pythium oligandrum M1 microorganism 5
x 105 per 1 ml
Properties:
Stability: 6 months at a temperature of up to 25 C.
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Example 3
Liquid biological antifungal product intended for the seed coating
Preparation:
Biomass obtained through culturing of the Pythium oligandrum M1 microorganism
on a
solid substrate was mixed in a homogenizer vessel (industrial mixer) with
demineralized water and
homogenized. Homogenization proceeded at 3,000¨ 5,000 revolutions per minute
for a period of 3
minutes. Subsequently, an osmolyte (sucrose) was added to the initial,
undiluted suspension in a
quantity corresponding to the resultant concentration by weight of 60 % weight
and to the required
number of oospores. The suspension with osmolyte (sucrose) was thereafter
homogenized in a
mixer for 1 minute.
It was more beneficial to prepare the product by combining the steps of
homogenization in
demineralized water and subsequent addition of osmolyte (sucrose) in one, in
such a way that the
biomass obtained through solid cultivation was homogenized in the vessel of an
industrial
homogenizer in an osmolyte solution (65 % sucrose solution). Homogenization
proceeded at 3,000
¨5,000 revolutions per minute for a period of 3 minutes. After determining the
number of oospores
in the initial suspension, this suspension was diluted with osmolyte (65 %
sucrose solution) in the
standard way such that the required concentration of oospores in the product
was achieved. The
suspension was then stored in sterile, stainless steel tanks at a temperature
of under 8 C.
The suspension obtained was then stored in sterile, stainless steel tanks at a
temperature of
under 8 C. The number of dormant oospores was determined microscopically with
the use of a
Sedgewick-Rafter counting chamber and stability was measured based on
microscopic observation
of the number of viable oospores using the plasmolytic method, in accordance
with the publication
by Etxeberria et al [13] from 2011. If the number of viable oospores did not
fall below 90 % of the
initial value, the product was marked as stable.
Stability: 6 months at a temperature of up to 25 C
Composition:
Sucrose as stabilizer 60
% weight
Water as stabilizer 36
% weight
Culturing biomass 4 %
weight
Number of dormant oospores of the Pythium oligandrum MI microorganism 2.5 x
106 per 1 ml
Example 4
Liquid biological antifungal product as a suspension concentrate with low
water content
Preparation:
Pythiurn oligandrurn M1 biomass from liquid cultivation was centrifuged
following
harvest. The supernatant was removed after centrifuging and the centrifuged
material was
supplemented with paraffin oil so that the resultant number of oospores was
500,000 oospores per I
ml of product. The mixture was thereafter homogenized in an industrial mixer
for 3 minutes. The
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suspension was stored in sterile containers at a temperature of under 8 C.
The number of dormant
oospores was determined microscopically with the use of a Sedgewick-Rafter
counting chamber
and stability was measured based on microscopic observation of the number of
viable oosporcs
using the plasmolytic method, in accordance with the publication by Etxeberria
et al [13] from
2011. If the number of viable oospores did not fall below 90 % of the initial
value, the product was
marked as stable.
Composition:
Culturing biomass
0.1 % weight
Paraffin oil as stabilizer
99.9 % weight
Number of dormant oospores of the Pythium oligandrum MI microorganism 5 x
105 per I ml
Properties:
Stability: 6 months at a temperature of up to 25 C.
Example 5
Liquid biological antifungal product applied in the form of nanofibers
constituting a slow-
release degradable matrix for the Pythium oligandrum M1 microorganism
Preparation:
Pythium oligandrum biomass from liquid cultivation was centrifuged following
harvest. The
supernatant was removed and the centrifuged material was supplemented with
osmolyte (65 %
sucrose) and biopolymer, which was thereafter processed using the technology
of electrostatic
spinning with the use of the non-toxic, biodegradable polymer polyvinyl
alcohol (PVA). The
number of dormant oospores was determined microscopically with the use of a
Sedgewick-Rafter
counting chamber and stability was measured based on microscopic observation
of the number of
viable oospores using the plasmolytic method, in accordance with the
publication by Etxeberria et
al [13] from 2011. If the number of viable oospores did not fall below 90 % of
the initial value, the
product was marked as stable.
Composition:
Main ingredient (liquid suspension of Pythium oligandrum microorganism) 84 %
weight
of which:
Sucrose as stabilizer 63 A
weight
Water as stabilizer 36
% weight
Culturing biomass 1 % weight
2. PVA carrier
PVA as degradable carrier 16
')/0 weight
Number of dormant oospores of the Pythium oligandrum Ml microorganism 5. 1
06 per 1 ml
Properties:
Stability: 6 months at a temperature of up to 25 C.
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Example 6
Liquid biological antifungal product applied in the form of impregnating
coating or spray
Preparation:
Pythiutn oligandrunt M1 biomass from liquid cultivation was homogenized after
harvest and
filtered using the filtering technique of separating particles with a size of
up to 300 m. After
centrifuging, this prepared biomass was mixed with mineral oil of low
viscosity such that the
required number of oospores in 1 ml of product was achieved. The number of
dormant oospores
was determined microscopically with the use of a Sedgewick-Rafter counting
chamber and stability
was measured based on microscopic observation of the number of viable oospores
using the
plasmolytic method, in accordance with the publication by Etxeberria et al
[13] from 2011. If the
number of viable oospores did not fall below 90 % of the initial value, the
product was marked as
stable.
Composition:
Culturing biomass 0.1 % weight
Mineral oil as stabilizer 99.9 % weight
Number of dormant oospores of the Pythiunt oligandrum Ml microorganism 2 x
105 per I ml
Properties:
Stability: 6 months at a temperature of 10 ¨ 45 C (depending on the type of
secondary substance).
Example 7
Liquid biological antifungal product applied as a sanitation product for air-
conditioning
Preparation:
Pythium oligandrunt M1 biomass from liquid culturing was homogenized after
harvest and
filtered using the filtering technique of separating particles with a size of
up to 100 - ,m. After
centrifuging, the biomass was diluted with a salt solution to the required
number of oospores per 1
ml of product. The principle of application is rinsing or spraying the filters
or piping of air-
conditioning or cooling units. The presence of the Pythiunt oligandrunt MI
microorganism
guarantees resistance to molds and fungal diseases.
Cleaning pollen filters:
The filter was submerged in a sanitary solution and was left in this for a
period of 10 ¨ 30
minutes, then rinsed. The alternative is to spray the same solution on the
filter in its operating
position. Dirt and remnants of the agent will be blown out by compressed air.
Sanitation of vaporizers and filters:
After cleaning vaporizers and/or filters, a solution of the product is
applied, without rinsing.
An excellent sanitation effect is achieved even when the product is only used
as a washing
detergent. The number of dormant oospores was determined microscopically with
the use of a
Sedgewick-Rafter counting chamber and stability was measured based on
microscopic observation
of the number of viable oospores using the plasmolytic method, in accordance
with the publication
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by Etxeberria et at [13] from 2011. If the number of viable oospores did not
fall below 90% of the
initial value, the product was marked as stable.
Composition:
Culturing biomass
0.1 % weight
Salt solution as stabilizer 99.9 %
weight
Number of dormant oospores of the Pythium oligandrum MI microorganism 5
x 105 per 1 ml
Properties:
Stability: 6 months at a temperature of 10 -- 45 C (depending on the type of
secondary substance).
Example 8
Liquid biological antifungal product applied as a sanitation product for the
treatment of
sediments ¨ for mud deposits and flooded areas
Preparation:
Pythium oligandrum M1 biomass from liquid cultivation was homogenized after
harvest
and filtered using the filtering technique of separating particles with a size
of up to 400 m. After
centrifuging, the biomass was diluted using distilled or demineralized water
to the required number
of oospores per 1 ml of product. The principle of action is the creation of a
water suspension
according to the dilution specified below and application in areas affected by
contaminated
deposits of mud and sediment, as a consolidation and improvement procedure to
improve the
properties of soils and earth. The number of dormant oospores was determined
microscopically
with the use of a Sedgewick-Rafter counting chamber and stability was measured
based on
microscopic observation of the number of viable oospores using the plasmolytic
method, in
accordance with the publication by Etxeberria et al [13] from 2011. If the
number of viable
oospores did not fall below 90 % of the initial value, the product was marked
as stable.
Composition:
Culturing biomass
0.1 % weight
Distilled water as stabilizer
99.9 % weight
Number of dormant oospores of the Pythium oligandrum M 1 microorganism 5
x 105 per 1 ml
The applicable utility is from 250 g per hectare at a concentration of 500,000
oospores per ml.
Properties:
Stability: 6 months at a temperature of up to 25 C.
Example 9
Liquid biological antifungal product as a stabilized aqueous suspension of
oospores of
Pythium oligandrum M1
Preparation:
Pythium oligandrum MI biomass obtained by liquid cultivation is mixed with
sterile
distilled water and homogenized after centrifuging (4,000 revolutions per
minute, 5 minutes) and
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removal of the supernatant. Homogenization proceeds at a high rate of
revolutions (20,000
revolutions per minute) for a period of I minute. Thereafter, the suspension
can be filtered and
concentrated as required for the purpose of achieving a suspension concentrate
containing the
required number of oospores of Pythium oligandrum Ml in 1 ml, without the
presence of remnants
of the original medium or remnants of mycelium. The suspension material
obtained can be stored
in sterile tanks at a temperature of between 1 and 8 C. The number of dormant
oospores was
determined microscopically with the use of a Sedgewick-Rafter counting chamber
and stability was
measured based on microscopic observation of the number of viable oospores
using the plasmolytic
method, in accordance with the publication by Etxeberria et al [13] from 2011.
If the number of
viable oospores did not fall below 90 % of the initial value, the product was
marked as stable.
Composition:
Sterile distilled water as stabilizer
99.9 % weight
Culturing biomass
0.1 % weight
Number of dormant oospores of the Pythium oligandrum M1 microorganism 5
x 10c per 1 ml
Properties:
Stability: a minimum of 6 months at a temperature of up to 25 C.
Example 10
Liquid biological antifungal product as a stabilized aqueous suspension with a
high
concentration of oospores of Pythium oligandrum M1
Preparation:
Pythium oligandrum M1 biomass obtained through liquid cultivation was
homogenized in a
homogenizer vessel (industrial mixer). Homogenization proceeded at 20,000
revolutions per
minute for a period of 3 minutes. The suspension was then centrifuged and,
after the supernatant
had been removed, the biomass was concentrated to the required number of
oospores in 1 ml of
product by mixing with osmolyte (65 % sucrose). The suspension concentrate was
stored in sterile,
stainless steel tanks at a temperature of under 8 C. The number of dormant
oospores was
determined microscopically with the use of a Sedgewick-Rafter counting chamber
and stability was
measured based on microscopic observation of the number of viable oospores
using the plasmolytic
method, in accordance with the publication by Etxeberria et al [13] from 2011.
If the number of
viable oospores did not fall below 90 % of the initial value, the product was
marked as stable.
Composition:
Sucrose as stabilizer
60 % weight
Water as stabilizer
35 % weight
Culturing biomass 5 %
weight
Number of dormant oospores of the Pythium oligandrum M1 microorganism
2.107 per 1 ml
Properties:
Stability: 6 months at a temperature of up to 25 C.
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Example 11
Biological antifungal product in the form of a paste on a base of glycerol
Preparation:
1197.1 g of glycerol of pharmaceutical quality (Glycerol 99 % PharmEur) was
poured into a
laboratory homogenizer and 239.4 g of silicon oxide SidentCD 22 S, 43.1 g of
culturing biomass,
14.4 g of vitamin E and 6 g of sage aroma were carefully sprinkled in. The
mixture was mixed at
60 revolutions per minute for a period of 10 minutes at laboratory
temperature. The mixture was
thereafter put into plastic tubes with stopper, 75 g at a time, with a total
of 20 such tubes being
filled in this way. The number of dormant oospores was determined
microscopically with the use of
a Sedgewick-Rafter counting chamber and stability was measured based on
microscopic
observation of the number of viable oospores using the plasmolytic method, in
accordance with the
publication by Etxeberria et al [13] from 2011. If the number of viable
oospores did not fall below
90 % of the initial value, the product was marked as stable.
Composition:
Glycerol 99 % Pharm Eur as stabilizer 79.77 %
weight
SiO2Sidente 22 S as filling on a base of silicon oxide
16.00% weight
Culturing biomass Pythium oligandrum M1
2.87 % weight
Vitamin E acetate as application material
0.96 % weight
FCO2 Bio 35 % sage aroma as aroma
0.40 % weight
Number of dormant oospores of the Pythium oligandrum Ml microorganism 2.5 x
104 per I ml
Properties:
Stability: a minimum of 6 months at a temperature of 25 C.
Example 12
Biological antifungal product in the form of a paste on a base of olive oil
Preparation:
600 g of virgin olive oil was poured into a laboratory homogenizer and 120 g
of silicon
oxide Sident 22 S, 21.6 g of culturing biomass, 7.2 g of vitamin E and 3 g of
mint aroma were
carefully sprinkled in. The mixture was mixed at 20 revolutions per minute for
a period of 10
minutes at laboratory temperature. The mixture was thereafter put into plastic
tubes with stopper.
75 g at a time, with a total of 10 such tubes being filled in this way. The
number of dormant
oospores was determined microscopically with the use of a Sedgewick-Rafter
counting chamber
and stability was measured based on microscopic observation of the number of
viable oospores
using the plasmolytic method, in accordance with the publication by Etxeberria
et al [13] from
2011.
If the number of viable oospores did not fall below 90 % of the initial value,
the product was
marked as stable.
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Composition:
Refined olive oil as stabilizer 79.77 % weight
SiO2 Sident 22 S as filling on a base of silicon oxide 16.00 % weight
Culturing biomass Pythium oligandrum M1
2.87 % weight
Vitamin E acetate as application material 0.96 %
weight
HNA peppermint essential oil as aroma
0.40 % weight
Number of dormant oospores of the Pythium oligandrum M1 microorganism 2.5 x
104 per 1 ml
Properties:
Stability: 6 months at a temperature of 25 C.
Example 13
Liquid biological antifungal product for the protection of aquatic organisms
The product is designed for the preventative protection of fish and young fish
(carp, roach,
bream) from a range of fungal diseases (in particular Aspergillus spp.). The
product is applied
preventatively in breeding tanks. The product is also intended for the
preventative protection of
fish eggs from attack by a range of fungal diseases (in particular Fusarium
spp., Aspergillus spp.).
Preparation:
Pythium oligandrum M1 biomass obtained through liquid cultivation was
homogenized in a
homogenizer vessel (industrial mixer). Homogenization proceeded at 20,000
revolutions per
minute for a period of 3 minutes. The suspension was then centrifuged and,
after the supernatant
had been removed, the biomass was concentrated to the required number of
oospores in 1 ml of
product by mixing with osmolyte (65 % sucrose). The suspension was stored in
sterile, stainless
steel tanks at a temperature of under 8 C. The number of dormant oospores was
determined
microscopically with the use of a Sedgewick-Rafter counting chamber and
stability was measured
based on microscopic observation of the number of viable oospores using the
plasmolytic method,
in accordance with the publication by Etxeberria et al [13] from 2011. If the
number of viable
oospores did not fall below 90 % of the initial value, the product was marked
as stable.
Composition:
Sucrose as stabilizer 63
% weight
Water as stabilizer 36 %
weight
Culturing biomass 1
% weight
Number of dormant oospores of the Pythium oligandrum M1 microorganism
5.10' per 1 ml
Properties:
Stability: 6 months at a temperature of up to 25 C.
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Example 14
Liquid biological antifungal product for treating honeycombs
The product is designed for the preventative treatment of honeycombs from
attack by a wide
range of funguses, in particular of the Aspergillus and Fusarium families. The
product can be used
to treat combs intended for storage outside bee hives and combs within a bee
hive not occupied by
bees.
Preparation:
Pythium oligandrum M1 biomass obtained by liquid cultivation was mixed in the
vessel of a
homogenizer (industrial mixer) with osmolyte (sucrose) and homogenized in such
a way that the
required number of oospores and concentration of osmolyte (sucrose) were
achieved.
Homogenization proceeded at 20,000 revolutions per minute for a period of 3
minutes. The
suspension was then stored in sterile, stainless steel tanks at a temperature
of under 8 C. The
number of dormant oospores was determined microscopically with the use of a
Sedgewick-Rafter
counting chamber and stability was measured based on microscopic observation
of the number of
viable oospores using the plasmolytic method, in accordance with the
publication by Etxeberria et
al [13] from 2011. If the number of viable oospores did not fall below 90% of
the initial value, the
product was marked as stable.
Composition:
Sucrose as stabilizer 64
% weight
Water as stabilizer 35.9 % weight
Culturing biomass
0.1 % weight
Number of dormant oospores of the Pythium oligandrum M1 microorganism
1.106 per 1 ml
Properties:
Stability: 6 months at a temperature of up to 25 C.
Example 15
Liquid biological antifungal product as an anhydrous suspension concentrate
intended for
spraying crops
Biomass obtained through cultivation of the Pythium oligandrum Ml
microorganism on a
solid substrate was mixed in a homogenizer vessel (industrial mixer) with
demineralized water and
homogenized. Homogenization proceeded at 3,000 ¨ 5,000 revolutions per minute
for a period of 3
minutes. After homogenization, the suspension obtained was mixed with an
inorganic carrier
(Sipernat), dried and ground in a cutting mill to achieve a resultant particle
size of up to 350 Rm.
The dry culturing biomass with carrier was then mixed with paraffin oil so as
to achieve the
required concentration of oospores in the product. The suspension was stored
in sterile containers
at a temperature of up to 25 C.
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The advantage of this product over example one is the longer stability of the
product, which
is ensured by the presence of water. The inorganic carrier, on a base of
silicon oxide, ensures good
suspensibility of solid particles (biomass) in the product.
The number of dormant oospores was determined microscopically with the use of
a
Sedgewick-Rafter counting chamber and stability was determined based on
microscopic
observation of the number of viable oospores using the plasmolytic method, in
accordance with the
publication by Etxeberria et al [13] from 2011.
Composition:
Culturing biomass
1.5 % weight
Sipernat 22 as inorganic carrier 6.5 % weight
Paraffin oil as stabilizer
92,0 % weight
Number of dormant oospores of the Pythium oligandrum MI microorganism
1.106 per 1 ml
Properties:
Stability: 24 months at a temperature of up to 25 C.
Example 16
Liquid biological antifungal product as an anhydrous suspension concentrate
intended for
spraying crops in the organic production of agriculture products
Biomass obtained through cultivation of the Pythium oligandrum MI
microorganism on
a solid substrate was mixed in a homogenizer vessel (industrial mixer) with
demineralized water
and homogenized. Homogenization proceeded at 3,000 ¨ 5,000 revolutions per
minute for a period
of 3 minutes. After homogenization, the suspension obtained was mixed with an
inorganic carrier
(Sipernat), dried and ground in a cutting mill to achieve a resultant particle
size of up to 350 f.tm.
The dry culturing biomass with carrier was then mixed with sunflower oil so as
to achieve the
required concentration of oospores in the product. The suspension was stored
in sterile containers
at a temperature of up to 25 C.
The advantage of this product over example one is the longer stability of the
product,
which is ensured by the presence of water. The inorganic carrier, on a base of
silicon oxide, ensures
good suspensibility of solid particles (biomass) in the product.
The number of dormant oospores was determined microscopically with the use of
a
Sedgewick-Rafter counting chamber and stability was determined based on
microscopic
observation of the number of viable oospores using the plasmolytic method, in
accordance with the
publication by Etxeberria et al [13] from 2011.
Composition:
Culturing biomass 1.5 %
weight
Sipernat 22 as inorganic carrier 6.5 % weight
Sunflower oil as stabilizer
92,0 % weight
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Number of dormant oospores of the Pythium oligandrum Ml microorganism 1.106
per 1 ml
Properties:
Stability: 24 months at a temperature of up to 25 C.
Example 17
Liquid biological antifungal product with reduced concentration of oospores
Pythium oligandrum M1 biomass obtained by liquid cultivation was mixed in the
vessel of
a homogenizer (industrial mixer) with osmolyte (sucrose) and homogenized in
such a way that the
required number of oospores and concentration of osmolyte (sucrose) were
achieved.
Homogenization proceeded at 20,000 revolutions per minute for a period of 3
minutes. The
suspension was thereafter mixed with osmolyte (65 % sucrose) such that the
concentration of
oospores in the product matched the requirements of the product. The
suspension was then stored
in sterile, stainless steel tanks at a temperature of under 8 C.
The number of dormant oospores was determined microscopically with the use of
a
Sedgewick-Rafter counting chamber and stability was measured based on
microscopic observation
of the number of viable oospores using the plasmolytic method, in accordance
with the publication
by Etxeberria et al [13] from 2011. The number of dormant oospores is
stabilized in the standard
way if required.
Composition:
Sucrose as stabilizer 64.95 % weight
Water as stabilizer 35,0 % weight
Culturing biomass 0.05 % weight
Number of dormant oospores of the Pythium oligandrum M1 microorganism 1 x
10 per 1 ml
Properties:
Stability: 12 months at temperatures of from 2 to 8 C; 6 months at a
temperature of up to 25 C.
Example 18
Laboratory test of the effectiveness of liquid biological antifungal products
in mycoparasitic
experiments
A laboratory test of the proliferation of Pythium oligandrum M1 from dormant
oospores of a
suspension concentrate according to exemplary embodiment 1 and of subsequent
mycoparasitism
on the phytopathogenic fungus Fusarium graminearum was carried out on sterile
agar plates with
nutrient medium on a base of malt extract. The diluted concentrate was applied
to an agar plate at
application level, meaning 0.2 ml of concentrate per 1 liter of water, and
part of this plate was
removed after sprouting and transferred to a different agar, which was
simultaneously inoculated
with Fusarium graminearum fungus.
The result of this test is presented in Figure 1, which shows a significant
discontinuation of
the growth of the phytopathogenic fungus Fusarium graminearum after the
addition of the Pythium
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oligandrum MI microorganism in comparison with standard growth (Figure 1A).
This situation is
further illustrated using a series of Petri dishes photographed after 4 days,
showing the joint growth
of both microorganisms on the one hand and the growth of the phytopathogenic
fungus on the other
(Figure 1B). After 11 days of culturing, significant suppression of the
phytopathogenic fungus is
clear alongside the growth of the Pythium oligandrum MI microorganism. If the
number of viable
oospores did not fall below 90 % of the initial value, the product was marked
as stable.
Example 19
Antifungal effectiveness against phytopathogenic funguses demonstrated in a
laboratory
experiment with wheat
Crop (wheat) seeds were treated with a solution of SAVO - sodium chlorite
solution -
(1 ¨ 5 % solution) and sprouted on agar soil extract. After sprouting, the
shoots were again treated
with 1 % Sava solution and placed on a layer of phyto agar in a 250 ml
Erlenmeyer flask. Flasks
were inoculated with a piece of agar (5 mm) with the phytopathogen Fusarium
gram inearum or
with the Pythium oligandrum MI microorganism (dormant oospores in suspension
concentrate) of
application concentration of 0.2 ml per liter and volume of 0.2 ml in the
following versions: 1)
phytopathogen; 2) phytopathogen + Pythium oligandrum Ml; 3) only Pythium
oligundrum Ml:
control sample which was not inoculated. Flasks were sealed with a thin layer
of cellulose and
gauze. Cultivation proceeded in a room at a temperature of 24 C. The growth
of the colony of
phytopathogens and the Pythium oligandrum M1 microorganism was monitored, as
was the
condition of the plant.
The result of this laboratory experiment is presented in Figure 2. As far as
the addition of
the actual phytopathogen is concerned, its significant growth in the
surroundings of the growing
wheat is quite clear, as is the damage to the plant caused by the pathogen
(Figure 2A). If the
Pythium oligandrum MI microorganism was also added, in addition to the
phytopathogenic fungus,
the presence of the phytopathogen was not visible and the cultivated plant
grew well (figure 2B). A
control experiment carried out in the presence of the Pythium oligandrum M1
microorganism alone
showed normal growth of the wheat plant which was comparable with experiments
without the
addition of any microorganism (Figure 2C).
Example 20
Test of the effectiveness of a liquid biological antifungal product intended
for spraying on
crops according to exemplary embodiment 2 in a greenhouse experiment on
Brassica napus
rapeseed
Four sectors were used at the company greenhouse in uherce containing growing
Brassica
napus rapeseed, Lohana Cl variety, whereby application was commenced at BBCh
15-21. Loose
biofungicide with mycoparasitic microorganism Pythium oligandrum M1
(Polyversure) was
applied in the first sector, a new liquid fungicide according to exemplary
embodiment 1 of this
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invention in sector 2, a new liquid antifungal product according to exemplary
embodiment 2 of this
invention in sector 3 and a new liquid anti-fungal product according to
exemplary embodiment 4 of
this invention in sector four. The application concentration in all cases was
0.5 g per liter, or 0.5 ml
per liter, with dispersion of 100 ml of such concentrated product per 1 square
meter of treated area.
.. A collection point was marked in each sector for the collection of samples
encompassing the plant
and the soil between the rows. Photographic documentation and a reading of the
temperature in the
greenhouse at the time of collection were also taken during each collection.
The collected samples
were transferred to the laboratory within an hour and stored in a freezer at -
20 C until the time of
analysis. Samples were collected before the application of the antifungal
products and then I hour,
2 hours, 7 hours, 20 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8
days, 11 days, 14 days,
17 days and 20 days after application.
The laboratory processing of samples involved photographic documentation of
individual
plants, the separation of leaf and root, determination of the wet weight of
the sample,
homogenization of the sample in DNA extraction buffer, centrifugation and the
conduction of
molecular analyses. The quantitative determination of the presence of the
Pythium oligandrum M1
microorganism was carried out using the qPCR method with a probe targeted at
the ITS sequence,
while the quantitative determination of the DNA of rapeseed was conducted
using an identical
method with a probe targeted at the Fat(A)A sequence. The quantitative
determination included the
execution of 50 cycles of DNA amplification under standard conditions and the
calculation of the
concentration of target sequences using calibration curve. The level of fungal
infestation was
detected in a similar way based on standard protocol.
The results of the effectiveness test are presented in Table 1 below. The
effectiveness
achieved by the new liquid antifungal products according to this invention is
in all cases
comparable with or better than the effectiveness of the solid, loose product.
Foreign funguses were
always suppressed depending on the colonization and amplification of the
Pythium oligandrum M1
microorganism in the place of application. The low level of contamination by
funguses was
thereafter maintained even after reducing the concentration of the Pythium
oligandrum M1
microorganism at the end of the experiment. The dynamic of the population of
the Pythium
oligandrum M1 microorganism was monitored in this experiment only for a period
of 15 days,
resulting in the absence of any comment on long-term effects. Nonetheless,
from the perspective of
the antifungal effectiveness of the product there is no doubt that the ability
to suppress fungus is
significant and long-term: there was a reduction in the occurrence of such
funguses of
approximately one order 24 hours after application and a further reduction of
almost one order after
a further 24 hours.
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Table 1
Measurement of the content of the Pythium ollgandrurn M1 microorganism and
measurement of the content of fungus on the leaves of rapeseed in a greenhouse
experiment.
The results are shown as the numbers of detected diploid genomes per 1 Eg of
host DNA of
rapeseed.
Time of Sector 1 Sector 2 Sector 3 Sector 4
collection Pythium Fungus Pythium Fungus Pythium Fungus Pythium Fungus
(h)
0 8 x 102 1 x 106 7 x 102 1 x 106 8 x 102 2
x 106 6 x 102 2 x 106
2 2 x 104 8 x 105 1 x 104 1 x 106 1 x 104 1
x 106 2 x 104 1 x 106
20 2 x 105 1 x 105 2 x 105 2 x 105 2 x 105 3
x 105 2 x 105 2 x 105
26 5 x10 6 x 104 6 x 105 6 x 104 7 x 104 5
x 104 6 x 105 5 x 10'
44 lx 106 2 x 104 2 x 106 2 x 104 lx 106 2
x 104 2 x 106 1 x 10-1
92 8 x 105 1 x 103 8 x 105 2 x 103 7 x 105 1
x 103 7 x 105 3 x 103
140 1 x 104 5 x 102 3 x 104 6 x 102 4 x 104 6
x 102 4 x 104 5 x 102
212 1 x 104 5 x 102 2 x 104 5 x 102 2 x 104 7
x 102 2 x 104 6 x 102
356 8 x 103 6 x 102 1 x 104 4 x 102 1 x 104 5
x 102 1 x 104 4 x 102
Example 21
Test of the effectiveness of a liquid biological antifungal product intended
for spraying on
crops according to exemplary embodiment 1 in a field test on Triticum aestivum
wheat
The winter wheat Triticum aestivum was sown in October 2015 in the company
field close
to the village of Oherce u Loun. The antifungal product according to exemplary
embodiment I of
this invention was applied to the designated tracks in November 2015 using the
standard spraying
method in a quantity of 100 ml per hectare in accordance with agricultural
standards and approved
registration for the Polyversum- antifungal preparation for plant protection.
Samples were taken
from 4 designated collection points before the application and at different
numbers of days after
application, whereby the growing wheat plants and the soil in proximity to the
sample were
collected. The samples were frozen immediately after collection and stored in
sealed bags or test
tubes at -20 C until the time of analysis. Nucleic acids were extracted from
the samples collected
using a published method (Klimeg R, Suchanek M, Ma talkova L et al /2016/
Comparison of the
efficacy of treatment of dermatophytosis by chemical and biological
antifungals: soil
peronosporomycete Pythium oligandrum is as efficient as the antifungal
enilconazole in the guinea
pig model, Vet Dermatol, in print) and used for molecular analysis).
As far as the dynamic of the occurrence of the Pythium oligandru In MI
microorganism
following spraying with the liquid product according to exemplary embodiment 1
is concerned, the
standardized concentrations on plants and in the surrounding soil showed
similar dependence.
Whereas only individual cells per g of sample could be identified by qPCR
genetic test prior to the
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application, this number rose to hundreds of cells per g of sample following
the application of the
liquid product corresponding to the normal standardized quantity. There was
visible multiplication
of the Pythium oligandrum Ml microorganism under the experimental conditions
in place after 48
hours, whereby this positive trend was interrupted under the conditions of a
specific experiment by
a frost episode at the beginning of December 2015, after which the dynamic
population growth was
not restored (compare Figure 3A and Figure 3B).
A quantity of valuable information was obtained through an analysis of gene
expression
profiles using the RT-qPCR method conducted at the "peak" phase of the
experiment, with the
highest growth of the Pythium oligandrum Ml microorganism. The values of the
concentration of
transcripts showed fast reaction to the occurring fungal pathogens at the
first phase (significant
expression of gene for cellulase) followed by an increased occurrence of
zoospores (endoglucanase
expression). Only after 48 hours and 120 hours is the characteristic gene
marker of sporulation
heightened, primarily in the surrounding soil and roots (Figure 3C). At the
same time, it was
possible to observe a significant reduction in the relative content of fungus
48 hours after
application (Figure 3D) and this reduction reciprocally correlated with the
expression of the gene
for cellulose responsible for the degradation of surface saccharides in
funguses during the
mycoparasitic action of the Pythium oligandrum MI microorganism (Figure 3E).
Example 22
Effectiveness test of toothpastes in the oral biofilm model with mechanical
plaque removal
A laboratory test using the oral biofilm model with mechanical removal of
plaque was
conducted according to the publication of Verkaik MJ et al [14] from 2010 in
order to monitor the
effectiveness of prepared toothpastes. A version (B) of the above-published
protocol was used,
consisting of two-hour adhesion with subsequent growth overnight (16-hour
biofilm) and
mechanical cleaning. The effectiveness of glycerol toothpaste containing the
Pythium oligandrum
M1 microorganism and toothpastes with olive oil and the Pythium oligandrum M1
microorganism
in removing the microorganisms of dental plaque was compared with two types of
commonly-
available toothpastes (Odol and Enzycal) and, as a control, mechanical
cleaning with a toothbrush
without any paste (marked Control). Identical paste without any Pythium
oligandruni content and
without the use of silicon oxide as an abrasive was used in a control
experiment.
The outcome of this test is described in the table of results in Figure 2.
This showed that
paste according to exemplary embodiment 4 exhibited considerably higher
effectiveness in the
removal of dental biofilm and plaque than the alternative products used, even
among individuals
affected by periodontal disease (Figure 4A to Figure 4D). Whereas the pastes
commonly used until
now reduced the intensity of biofilm formation to around one half, the new
pastes according to this
invention to up to one tenth. Its effect in the control paste without any
Pythium oligandrum M1
microorganism content was comparable with common pastes, while the effect was
significantly
higher in the control paste without silicon oxide. The overall level of
biofilm formation in the
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patient with periodontal disease was significantly higher and it was also far
better to observe the
differences in the effectiveness of individual pastes.
Example 23
Test of the effectiveness of the liquid biological antifungal product
according to exemplary
embodiment 1 in protecting living spaces from fungal spores
The antifungal product according to exemplary embodiment 1 was diluted with
water to a
concentration of 330,000 oospores per liter of application solution, i.e. 0.66
ml of liquid
concentrate was added to 1 liter of solution. A control solution was also
prepared under identical
conditions, but without Pythium oligandruni M1 microorganism content; the
biomass was replaced
with water. The solutions prepared in this way were applied in two small,
enclosed rooms at the
farmstead at Oherce u Loun No. 2 in the form of spraying a fine mist using an
atomizer. Following
application, both rooms were impermeably closed and fallout tests were
conducted on the content
of spores using the Omeljanskji method. The results were evaluated in the
laboratory of Dr.
Miroslav Kolafik at the Institute of Microbiology of the Academy of Sciences
of the Czech
Republic in Prague.
The results of the experiment are presented below in Table 2. The experiment
was
conducted in closed rooms without any flow of air and for this reason the
observed changes are
slower and were observed only after several months. Nonetheless, it is clear
from the results that in
control room 1, with the application of auxiliary substances, the numbers of
spores identified
fluctuated, but did not at any time of observation fall below the WHO (World
Health Organization
limit) of 500 spores per 1 cubic meter of air. By contrast, there was a
statistically significant
decrease in spores 3 months after application in room 2 and this subsequently
fell below the WHO
limit 6 months following the date of application.
Table 2
Measurement of the number of fungal spores per 1 cubic meter in room 1, with
the
application of a control product, and in room 2, with the application of a
product containing
the active ingredient Pythium oligandrum M1
Months after 0 1 3 6
application (0 ¨
before
application)
Room 1, control 5000 4500 3500 4500
Room 2 5000 4000 1300 400
(product accord.
to exemplary
embodiment 1)
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Example 24
Method of production of a liquid biological antifungal product containing
Pythium
oligandrum M1
The detailed nature of the liquid biological antifungal product containing
Pythium
oligandrum MI according to this invention was demonstrated in detail in the
previous exemplary
embodiments.
Examples of the method of production of the liquid biological antifungal
product containing
Pythium oligandrum M1 according to this invention are presented below.
A medium containing extract of cereals, cane molasses and other essential
nutrients is used
for the aerobic cultivation of Pythium oligandrum M1 oomycetes. The liquid
medium is sterilized
in a steam sterilizer. After cooling, it is inoculated with one of the
selected strains of Pythium
oligandrum MI. After cultivation has ended, a process which takes
approximately 13 days, the
biomass is harvested and processed in the manner presented above in the
exemplary embodiments.
A medium is prepared for cultivation the Pythium oligandrum M1 microorganism
on a solid
substrate containing cereal grains, preferably hulled millet grains, for
example PalliCUI17 177iliClee14117
L., which are mixed with a percentage of nutrient liquid medium. The medium is
sterilized in
cultivation vessels in a steam sterilizer. After cooling, the medium is
inoculated with one of the
selected strains of Pythium oligandrum Ml. After the process of cultivation
has ended, a process
which takes approximately 8 days, the harvested biomass is further processed
into the form of a
suspension concentrate, as is described above in the exemplary embodiments.
After the end of the culturing stage of Pythium oligandrum in the liquid phase
and in solid
phase, the biomass is homogenized with the liquid medium, for example in an
industrial mixer, so
that a minimum of 95 % of the particles in the suspension are between 0.05 and
0.30 mm,
preferentially between 0.050 and 0.125 mm. The homogenous suspension is
subsequently
characterized by the number of oospores. The suspension is standardized ¨
concentrated or diluted
¨ according to the number of oospores to the pre-determined concentration of
oospores suitable for
the preparation of the liquid biological antifungal product at issue.
After the end of the culturing phase of Pythium oligandrum M1 on a solid
substrate, the
biomass is homogenized, preferentially with an industrial mixer, in the
corresponding volume of
demineralized water so that the resulting size of a minimum of 95 % of the
particles in the
suspension is between 0.050 and 0.300 mm in size, preferentially between 0.050
and 0.125 mm.
The homogenous suspension is subsequently characterized by the number of
oospores. The
suspension obtained is concentrated or diluted according to the number of
oospores to the pre-
determined concentration of oospores suitable for the preparation of the
liquid biological antifungal
product at issue.
The suspension is separated, for example filtered, after the phase of
homogenization and
concentration, or dilution, to ensure the maximum admissible size of particles
within the relevant
range. If achieving particles of around 0.05 mm to 0.125 mm, there is a high
probability of
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obtaining a concentrated suspension, or an almost pure concentrated suspension
of oospores of
Pythium oligandrum Ml. Most of the exemplary embodiments presented above had a
size of
particles of predominantly 0.050 to 0.125 mm.
An aqueous suspension may be stabilized during homogenization with an additive
of
osmolyte and can be stored in large, sterile containers at temperatures of
lower than 8 C.
In the case of an anhydrous suspension of the Pythium oligandrum Ml
microorganism, the
material obtained after the end of the culturing stage on a solid substrate or
in liquid phase is
homogenized, preferentially with an industrial mixer, so that the resulting
size of a minimum of 95
% of the particles in the suspension is between 0.050 and 0.300 mm,
preferentially between 0.050
and 0.125 mm. The homogenous suspension obtained, characterized by the number
of oospores, is
subsequently centrifuged and, after removal of the supernatant, oil is added
to the centrifuged
material to achieve the pre-determined concentration of oospores, as
appropriate for the preparation
of the liquid biological antifungal product at issue. The material obtained is
subsequently re-
suspended, for example using an industrial mixer.
Specific possible methods of production are specified in the exemplary
embodiments
presented above, but are not limited to these.
Industrial Applicability
The new liquid antifungal product containing a stabilized suspension of
oospores can be
made in sizeable volumes mainly because the fermentation production phase and
the formulation
stage are both well manageable from the technical perspective. The new liquid
product can find an
application in all areas in which other forms of microscopic oomycetes of
Pythium oligandrum
have been applied and used until now, in particular in the biological
protection of plants, to protect
buildings and residences from funguses, to suppress funguses and yeasts and to
establish
physiological balance of microflora in human and veterinary applications. Its
use as a protective
spray for plants with degradable polymer, acting as a protective factor and as
a slow-release,
degradable matrix "carrier" of the Pythium oligandrum microorganism, is highly
innovative. The
product according to this invention can also be beneficially used to coat or
spray wooden
structures, when the oil components of the product also lead to effective
impregnation of the wood.
The presence of the microorganism guarantees resistance to funguses and fungal
diseases and
"defects" to wood. One entirely new use is the effective conservation of air-
treatment equipment,
air-conditioning and cooling units. It would also appear advantageous to use a
product specially
designed for purpose to reduce the level of fungal contamination in areas
affected by flooding, in
dried-out areas and in the surroundings of recultivated areas of water.
30
